Effective control of systemic autoimmune diseases such as rheumatoid arthritis (RA) currently relies on the use of intense generalized immunosuppression, thus increasing the risk of infection and malignancy. A better theoretical approach to the treatment of autoimmune disease is the induction of an antigen-specific tolerance that targets only the dangerous self-reactive T and B cells. The design of such effective therapies with the potential to cure autoimmune disease, however, is hampered by our inability to visualize and study those self antigen-specific polyclonal lymphocytes within the intact immune system. Furthermore, we lack sufficient knowledge regarding the induction of clonal anergy in lymphocytes that are already responding to self antigens within the diseased individual. Finally, our typical candidate approach to the identification of new therapeutic targets is an inherently slow process that can be adversely influenced by preconceived notions about the molecular mechanisms that are important to the induction of anergy. We now propose a series of pre-clinical studies that will make use of emerging antigen/class II tetramer technologies to track polyclonal CD4+ T cells that break immune tolerance and cause autoimmunity. Specific Aim 1 will investigate the individual roles of NT5'E and Foxp3 in the development and maintenance of clonal anergy following initial self antigen encounter by polyclonal CD4+ T cells in the peripheral immune system. Ecto-5'-nucleotidase (NT5'E), a novel candidate anergy factor discovered using a genome-wide screen for chromatin modifications in association with changes in gene expression, will be evaluated for its role in the control of clonal anergy development and maintenance of antigen unresponsiveness. Specific Aim 2 will establish the role of clonal anergy in the epigenetic changes that protect against the development of CD4+ T cell-mediated arthritis. Experiments under this aim will model RA using an adoptive transfer of KRN TCR-transgenic T cells reactive to the natural autoantigen glucose-6-phosphate isomerase (GPI), and investigate in the setting of systemic inflammation or lymphopenia the roles of NT5'E and Foxp3+CD4+ T regulatory cells in the epigenetic control of clonal anergy induction. In addition to increasing our fundamental knowledge about the regulation of anergy induction by NT5'E and immunoregulation in vivo, the results obtained with the class II tetramers will also serve as a paradigm for the development of biomarkers for use in human trials designed to target autoreactive CD4+ T cells for clonal anergy induction. Furthermore, our ongoing investigation of the epigenetic control of gene expression during CD4+ T cell responses to self antigen offers the opportunity to identify additional novel therapeutic targets. PUBLIC HEALTH RELEVANCE: The development of a cure for diseases such as rheumatoid arthritis will require the design of therapies that can effectively restore immune self tolerance in T cells. To develop such therapies, we need to create tools that can monitor the state of tolerance in offending T cells during human trials. Furthermore, we need to discover additional molecular targets for therapeutic intervention that will promote tolerance induction in autoreactive T cells. This project will develop the appropriate tools to monitor the behavior of autoreactive T cells in several mouse models of autoimmunity, will test one potential therapeutic target (NT5'E) for its role in tolerance induction, and will take advantage of new gene array technology to discover additional therapeutic targets.